The present invention relates to the detection of enzymes of hydrolase type, in particular peptidases, by using effective chromogenic substrates. The present invention also relates to a process and a device for identifying microorganisms which are both simple and reliable.
Specific substrates have been used for many years to determine the presence or absence of enzymatic activities characteristic of bacteria. By the choice of substrates, depending on whether or not there is a reaction, it is possible to characterize the nature of a genus of bacteria or to differentiate between the species of a given bacterial genus.
Synthetic substrates of enzymes consist of two portions, a first portion which is specific for the enzymatic activity to be detected, and a second portion which acts as a label, and which is referred to herein-below as the labeling portion.
These specific substrates can be fluorescent or chromogenic substrates. In point of fact, it is the second portion or labeling portion which is fluorescent or chromogenic, when it is not combined with the first portion.
Fluorescent substrates may be of diverse composition.
First of all, substrates based on umbelliferone or aminocoumarin, and the derivatives thereof substituted in position 2, which allow the release of a fluorescent compound whose color ranges from blue to green under an ultraviolet (UV) lamp (xcexex=365 nm).
Next, substrates based on resorufin (and derivatives), in which there is release of a compound which is fluorescent pink under natural light (xcexex=530 nm).
Finally, substrates based on fluorescein (and derivatives) which, after degradation, releases a compound which is fluorescent yellow under natural light (xcexex=485 nm).
These substrates are unsuitable for use in agar media, and are used more in liquid medium.
The chromogenic substrates may also be of diverse nature.
Firstly, there are substrates based on indoxyl and its derivatives which, in the presence of oxygen, produce a precipitate ranging from blue to pink.
Their applications are essentially limited to osidases and esterases and do not concern the detection of a peptidase activity. Whereas they are well suited to use on a solid support (filter, agar, electrophoresis gel, etc.), they are less well suited to the use in liquid aqueous medium (formation of a precipitate).
Secondly, there are substrates based on hydroxyquinoline or esculetin and their derivatives, which produce a brown precipitate in the presence of iron salts.
In this case also, their applications are limited to osidases and esterases. They are suitable for use on a solid support, and relatively unsuitable for use in liquid aqueous medium.
Thirdly, there are substrates based on nitrophenol and nitroaniline and derivatives, which lead to the formation of a yellow compound.
They make it possible to detect osidase and esterase activities in the case of nitrophenol-based substrates, and peptidase activities in the case of nitroaniline-based substrates. However, in the case of detecting peptidase activities, the nitroaniline released is toxic to the bacteria which it is desired to identify or characterize, which may have a negative impact on current or subsequent analyses. Moreover, they are relatively unsuitable for use on a solid support, and are better suited to use in liquid medium. Furthermore, they are not particularly chromogenic on account of the relatively low extinction coefficient of the color (yellow) which gives a relatively weak contrast in biological media.
Fourthly, there are substrates based on naphthol and naphthylamine and its derivatives. In this case, the reaction is carried out in two stages; the naphthol or naphthylamine released by the enzymatic activity undergoes an xe2x80x9cazo-couplingxe2x80x9d in the presence of a diazonium salt which is added at the detection stage, leading to the formation of a colored insoluble compound.
They make it possible to detect osidase and esterase activities by means of naphthol, and peptidase activities by means of naphthylamine. The azo-coupling reaction is carried out in a medium which is often chemically corrosive and toxic to bacteria, making the sample unusable for other analyses, and, what is more, naphthylamines are carcinogenic.
To detect naphthylamine and thus a peptidase activity, it is also possible to add p-dimethylamino-cinnamaldehyde in acidic medium at the end of the enzymatic reaction, instead of a diazonium salt, although this still has toxicity drawbacks with respect to the sample analyzed.
Patent application FR-A-2 708 286 proposes the use of a mixture of chromogenic substrates, each chromogen giving a particular coloration for a specific enzyme which is different from the coloration and enzyme associated with the other chromogen. When the two colorations and thus the two enzymes are present, there is formation of a xe2x80x9ctertiary colorationxe2x80x9d.
However, this technique is unsatisfactory since, as a function of the low concentration of one of the two enzymes, it is not possible to detect this enzymatic activity which is thus masked by the coloration associated with the enzyme whose concentration is predominant.
Finally, patents U.S. Pat. No. 4,681,841 and U.S. Pat. No. 4,588,836 describe an indirect method for detecting a single enzymatic activity using a coupling between an aminobenzene and a hydroxyaromatic derivative (for example xcex1-naphthol), this coupling leading to the formation of a chromogenic indicator in the presence of oxidase. One of the two compounds (the aminobenzene) forms part of the composition of the starting substrate; if the desired enzymatic activity is present, this compound will be released and will be able to react with the other compound.
Nevertheless, in order for the detection of the desired enzyme to be possible, the presence of oxidase in the reaction medium is an absolute necessity. However, although this information discourages a person skilled in the art from looking for a solution which does not use oxidase, the Applicant has proved, by numerous tests carried out in its laboratories, that it is possible to detect the desired enzyme by means of using, for example, aminobenzene and xcex1-naphthol, without the addition of oxidase.
It may thus be readily appreciated that no chromogenic substrate which is particularly effective and advantageous as regards detecting at least one peptidase activity currently exists.
As regards the identification process and device, the state of the art consists of an identification process which involves a three-step manipulation:
taking a sample of the colony to be identified,
carrying out an orinetation test, and
looking for colonies similar to those observed and preparing an inoculum.
The orientation test should be performed before using an identification system. This is especially the case for Gram staining which requires an observation by microscope.
However, this coloration is not always easy to carry out and above all to interpret. Moreover, the cost of this test is far from negligible.
One of the aims of the present invention is thus to create a link between a culture medium and antibiotic assay and identification systems, offering biologist the possibility of performing a simple one-step test both for confirming the result of the Gram staining and for preparing the inoculum. Thus, the choice of antibiotic assay and identification tests is made reliable.
Patent application EP-A-0 122 028 proposes a colorimetric method for detecting the presence of at least one enzyme suspected of being present in a biological sample. It recommends the preparation of an absorbent brush, absorbing in this material at least one susbstrate which is specific for the enzyme which it is desired to detect. The absorbent material containing the substrate(s) is dried before use.
The invention allows the colormetric detection of enzymatic activities of hydrolase (osidase, esterase, phosphatase or peptidase) type with the aid of synthetic substrates based on two compounds, as well as a novel process which can be applied to both liquid and solid reaction media.
The invention also proposes a process and a device for identifying microorganisms, which orient the identification, and allow the recovery of said microorganisms in order to allow an inoculum to be prepared. This inoculum makes it possible to use the same microorganisms for one or more other steps, such as an identification or an antibiotic assay.
After hydrolyzing the substrates, the enzymatic activities are detected on the basis of the formation of a colored complex from the oxidative coupling of the two compounds mentioned above. This oxidative coupling can be facilitated by an oxidizing agent added to the reaction medium or produced during a metabolic process within this medium, or more simply by the presence of endogenous oxygen in this same medium.
According to a first interpretation, the present invention relates to a combination of two distinct entities, one being a nonchromogenic molecule and the other being the substrate for detecting the presence of the enzymatic activity or at least one enzyme. The substrate is characterized in that in consists of a specific portion of the enzyme, and of a nonchromogenic molecule other than the first molecule, which constitutes the labeling portion of the substrate; the two nonchromogenic molecules react together when they are in free form and can generate a chromogenic molecule.
Still according to this first interpretation, the invention also relates to a combination of two distinct entities, each corresponding to a different substrate and making it possible to detect the presence of the enzymatic activity of at least two enzymes. Each substrate is characterized in that it consists of a specific portion of the desired enzyme, and of a nonchromogenic molecule constituting the labeling portion of the substrate, and in that the two nonchromogenic molecules corresponding to the two substrates react together when they are in free form and can generate a chromogenic molecule.
According to a second interpretation, which will be used more particularly hereinbelow, to arrive at this result, it is necessary to use a new form of substrates. Thus, the substrate according to the invention comprises two different molecules. One of the molecules consists of a specific portion, an enzymatic activity and a labeling portion, which is distinct for a chromogenic molecule. The other molecule always comprises the labeling portion, distinct for a chromogenic molecule, which is free or associated with at least one specific portion, of an enzymatic activity. The object of the invention is thus, if the enzymatic activity(ies) is (are) present, to reveal the formation of a chromogenic molecule which is effective only when the two labeling portions, once released, have become associated.
To this end, the present invention relates to a chromogenic substrate for detecting the presence of the enzymatic activity of at least one enzyme, characterized in that it consists of at least two molecules, a first molecule consisting of a nonchromogenic labeling portion associated with at least one specific target portion of the enzyme and a second molecule consisting of another nonchromogenic substance, and in that the nonchromogenic labeling portion, once released, reacts with the second molecule to form a chromogenic molecule.
The present invention also relates to a chromogenic substrate for detecting the presence of the enzymatic activity of at least two enzymes, characterized in that it consists of at least two molecules, a first molecule consisting of a nonchromogenic labeling portion associated with at least one specific target portion of the first enzyme and a second molecule consisting of another nonchromogenic labeling portion associated with a specific target portion of the second enzyme, and in that the nonchromogenic labeling portions, once released, react to form a chromogenic molecule.
Irrespective of the method of preparation of the substrate, the nonchromogenic labeling portion of the first molecule consists of aminobenzene or a derivative thereof: 
that [sic] the nonchromogenic labeling portion of the second molecule consists of xcex1-naphthol or a derivative thereof: 
and that [sic] the chromogenic molecule obtained consists of: 
In this case, the radical R1 consists of xe2x80x94OH, xe2x80x94SH or 
the radical R10 consists of xe2x80x94H or an atom, such as xe2x80x94Br, xe2x80x94Cl or xe2x80x94I, or a group of atoms, such as xe2x80x94SH, which can be removed during the oxidative coupling, and each radical R2 to R9, R11, or R12 consists of xe2x80x94H, xe2x80x94OH, xe2x80x94Br, xe2x80x94Cl, xe2x80x94I or other more complex substituents, such as xe2x80x94CH3, xe2x80x94CH2CH3, xe2x80x94OCH3, xe2x80x94OCH2CH3 or xe2x80x94COOH.
In the case of the radical R1, X/or Z consists of xe2x80x94H, or other more complex substituents, such as xe2x80x94CH3, xe2x80x94CH2CH3, 
According to one embodiment, at least one of the pairs of radicals R2/R3 and R4/R5 consists of an aromatic, alicyclic or heterocyclic system.
The present invention also relates to a process for detecting an enzymatic activity via substrates, as described above, which consists in:
placing the substrates in contact with bacteria of at least one type,
waiting for the bacteria to hydrolyze the substrates, and
detecting the enzymatic activities based on the formation of a colored complex from the oxidative coupling of the two labeling portions.
Another object of the present invention is to propose a process, in which the molecules are present in an absorbent material and are placed in contact with the sample, and, after detection, the microorganisms thus withdrawn are resuspended in order to allow subsequent analyses (identifications, antibiotic assays, etc.).
The molecules and other compounds forming part of the reaction composition contained in the absorbent material are:
the first molecule consisting of aminobenzene or a derivative thereof, and
the second molecule consisting of xcex1-naphthol or a derivative thereof.
The reaction composition also contains an oxidizing agent, such as potassium ferricyanide.
Among the other compounds, it is also possible to have a reaction activator, such as a small amount of the first molecule and/or of the second molecule, which is (are) present in the sample to be tested.
Still among the other compounds, it is possible for the composition to comprise a binder or adhesive, such as PolyVinylPyrrolidone (PVP).
The process can be used for the Gram identification of a bacterial species to be tested and, in this case, the first molecule consists of AlaDMpPD and that [sic] the second molecule consists of xcex1-naphthol.
Furthermore, the composition of the reaction mixture absorbed by the absorbent material is as follows:
xcex1-naphthol from 0.01 g/l to 5 g/l, preferably from 0.1 g/l to 1 g/l, and for example 0.5 g/l,
potassium ferricyanide from 0.01 g/l to 5 g/l, preferably from 0.1 g/l to 1 g/l, and for example 0.5 g/l, and
AlaDMpPd from 0.01 g/l to 5 g/l, preferably from 0.1 g/l to 1 g/l, and for example 0.35 g/l.
According to one variant, the activator consists of Ala-DMpPD at a concentration of from 0.01 g/l to 0.5 g/l, preferably from 0.05 g/l to 0.1 g/l, and for example 0.075 g/l.
According to another variant, the composition also comprises from 1 g/l to 50 g/l, preferably from 10 g/l to 25 g/l, and for example 15 g/l, of PVP.
The invention also relates to the use of substrates, as described above, for detecting an enzymatic activity of peptidase type.
Finally, the present invention relates to an identification device for carrying out the identification process described above, which consists of a support, for example made of plastic, which is inert with respect to the absorbent material and to the substrate(s) it contains and/or with respect to the test sample, on which is mounted a head made of an absorbent material, such as viscose.
I-Novel chromogenic substrates and implementation process:
The invention, which will be described below, relates to a few specific embodiments of the invention. These embodiments therefore do not limit the scope of the present invention which can be used for detecting enzymatic activities of any type, of hydrolase type, and for microorganisms of any genus or type.
The chromogenic substrate for detecting the presence of the enzymatic activity of at least one enzyme consists of at least two molecules, a first molecule consisting of a nonchromogenic labeling portion associated with at least one specific target portion of the enzyme and a second molecule consisting of another nonchromogenic substance. The nonchromogenic labeling portion, once released, reacts with the second molecule to form a chromogenic molecule.
In another case, the chromogenic substrate for detecting the presence of the enzymatic activity of at least two enzymes consists of at least two molecules, a first molecule consisting of a nonchromogenic labeling portion associated with at least one specific target portion of the first enzyme and a second molecule consisting of another nonchromogenic labeling portion associated with a specific target portion of the second enzyme. Once released, the nonchromogenic labeling portions react to form a chromogenic molecule.
In the case of the detection of only one enzymatic activity, there are two nonchromogenic molecules, i.e. a first molecule having a portion which is specific for the enzymatic activity to be detected and a labeling portion, whereas the second molecule consists only of the labeling portion. The two labeling portions consist respectively of compounds I and II below: 
in which:
R1=xe2x80x94OH or xe2x80x94SH or 
xe2x80x83with X and/or Z=xe2x80x94H or other more complex substituents, such as xe2x80x94CH3, xe2x80x94CH2CH3, 
R10=xe2x80x94H or a group or atom which can be removed during the oxidative coupling, such as xe2x80x94Br, xe2x80x94Cl, xe2x80x94I, xe2x80x94SH, etc.,
R2, R3, R4, R5, R7, R8, R9, R11 and R12=xe2x80x94H, xe2x80x94OH, xe2x80x94Br, xe2x80x94Cl, xe2x80x94I, xe2x80x94CH3, xe2x80x94CH2CH3, xe2x80x94OCH3, xe2x80x94OCH2CH3, xe2x80x94COOH, or any other more complex substituent, R2/R3 and R4/R5 also possibly forming part of an aromatic, alicyclic or even heterocyclic system, such as: 
Compound III obtained by combining the two compounds I and II is thus as follows: 
To understand the invention, it is recalled that the formula of a hydrolase enzyme substrate may be represented schematically in the form:
Axe2x80x94Oxe2x80x94B, if it is an osidase, phosphatase, esterase or sulfatase substrate.
The enzymatic hydrolysis may then be written as:
Axe2x80x94Oxe2x80x94B+H2Oxe2x86x92Axe2x80x94OH+Bxe2x80x94OH
with, according to the invention:
Axe2x80x94OH=ose, phosphate, sulfate, fatty acid (from the simplest, i.e. acetic acid), and
Bxe2x80x94OH=compound I with R1=xe2x80x94OH or compound II.
Axe2x80x94COxe2x80x94NHxe2x80x94B, if it is a peptidase substrate.
The enzymatic hydrolysis can then be written as:
Axe2x80x94COxe2x80x94NHxe2x80x94B+H2Oxe2x86x92Axe2x80x94COOH+Bxe2x80x94NH2,
with, according to the invention:
Axe2x80x94COOH=amino acid or chain of amino acids possibly ending with a blocking agent, and
Bxe2x80x94NH2=compound I.
The process according to the invention uses, on the one hand, an enzymatic substrate as described above, based on:
either a compound I derived from aminobenzene, with R1=xe2x80x94OH for the osidase, phosphatase or esterase substrates,
or a compound II derived from xcex1-naphthol (with the exclusion of peptidase substrates), on the other hand, a revealing agent which is initially present in the reaction medium or which can be added during or after the enzymatic reaction, this agent being:
either compound II for the detection of compound I resulting from the enzymatic hydrolysis of the substrates based on the latter compound, and
or compound I for the detection of compound II resulting from the enzymatic hydrolysis of the substrates based on the latter compound.
The formation in the reaction medium of the colored compound III (derived from indophenol) from the oxidative coupling of I and II makes it possible to detect the hydrolysis of the substrate and thus the enzymatic activity involved.
The color of compound III depends on the substituents on compounds I and II.
For example, it is purple if R1=xe2x80x94OH and R2 to R11=xe2x80x94H, or blue if R1=xe2x80x94OH, R2 and R5=xe2x80x94Cl and R3 to R11=xe2x80x94H.
The process thus described is, in particular, extremely advantageous for calorimetrically detecting activities of peptidase type. Specifically, according to the prior art, the choice of colored reactions was limited to the use of substrates based on nitroaniline, which is relatively nonchromogenic, and of substrates based on naphthylamine, which is highly toxic, necessitating the addition of a reagent at the end of the reaction (for example a diazonium salt) to detect the amine released.
The invention proposes a novel type of substrates for peptidases, based on aminobenzene or derivatives (compound I).
Gradually as this compound I is released into the reaction medium during the enzymatic hydrolysis, it forms a strongly colored complex (generally colored red to blue) by oxidative coupling with xcex1-naphthol or a derivative (compound II) present in this medium.
The coupling reaction can also be obtained at the end of the test if the addition of compound II is carried out at the end of the enzymatic reaction.
Another important advantage of the invention consists in combining two substrates in the reaction medium, according to the process, one based on compound I and the other based on compound II.
Any type of combination of simultaneous detection of two enzymatic activities (for example a peptidase and an osidase, an esterase and an osidase, two osidases, etc.) may thus be envisaged. In these cases, the colored complex III will be formed only if the two substrates are hydrolyzed. This combination may be very useful for diagnostic tests which require high specificity, in particular when it is a case of characterizing microorganisms.
One of the examples mentioned illustrates the advantage of this simultaneous detection of two enzymatic activities in the field of identifying bacteria.
Among the other advantages of the invention, it is possible to give compound I a double functionality. Specifically, it is possible to chemically graft onto compound I, on the one hand onto the xe2x80x94NH2 group, an amino acid or a chain of amino acids possibly ending with a blocking agent, and, on the other hand, onto R1, if it is a hydroxyl (xe2x80x94OH) group, an ose, a phosphate or a fatty acid. A molecule is obtained which may be termed a double substrate and which, in order to generate compound I in free form in the reaction medium, should be hydrolyzed by two distinct enzymatic activities, on the one hand a peptidase, and on the other hand an osidase, phosphatase or esterase, depending on the nature of the product grafted onto R1.
Thus, in the presence of compound II, the colored complex III can only be obtained if the two enzymatic activities are present. The advantage of such substrates with double functionality lies in their very high specificity which may be exploited to characterize microorganisms, for example.
In the same sense, a mixture of a substrate with double functionality based on compound I, as described above, and a substrate based on compound II may be envisaged according to the invention to search for another osidase, phosphatase or esterase. In this case, the production of the colored complex III requires the simultaneous presence of three enzymatic activities, in order to release compounds I and II. Two enzymatic activities make it possible to hydrolyze the molecule based on compound I and one enzymatic activity makes it possible to hydrolyze the molecule based on compound II. The specificity of the test is thus further increased.
According to the invention, it is also possible to combine different types of substrates in the same reaction medium, in particular substrates known in the prior art and substrates according to the process claimed. This makes it possible to carry out different colored reactions, and thus to simultaneously detect several enzymatic activities in the same reaction medium.
The invention can be applied to the search for enzymatic activities in different types of biological or nonbiological samples.
The invention can also be used to characterize microorganisms such as bacteria or yeasts. In this case, the enzymatic tests can be performed in liquid medium in individual tubes or in compartmentalized supports, such as microtitration plates, for example. They can also be performed in agar medium (for example in Petri dishes) with staining of the colonies producing the desired enzymatic activities.
The present invention thus relates to a process for detecting an enzymatic activity by means of substrates, as described above, which consists in:
placing the substrates in contact with bacteria of at least one type,
waiting for the bacteria to hydrolyze the substrates, and
detecting the enzymatic activities based on the formation of a colored complex from the oxidative coupling of the two labeling portions.
The oxidative coupling is facilitated either by:
the addition of at least one oxidizing agent to the reaction medium,
the production, during a metabolic process in this reaction medium, of at least one oxidizing agent,
the presence of endogenous oxygen in said reaction medium.